THE AGONIST BINDING-SITE OF THE GAMMA-AMINOBUTYRIC-ACID TYPE-A CHANNEL IS NOT FORMED BY THE EXTRACELLULAR CYSTEINE LOOP

The amino-terminal extracellular domain of the subunits comprising the gamma-aminobutyric acid (GABA) receptor contains two cysteine residue s (designated at relative positions 1 and 15) separated by 13 amino ac ids. These two cysteines (presumably disulfide bonded) are located app roximately 150 amino acids from the amino terminus. There is significa nt homology in the amino acid sequence of this cysteine loop both betw een the different subunits of the GABA receptor and with subunits of o ther members of this ligand-gated ion channel superfamily (nicotinic a cetylcholine- and glycine-activated ion channels). A number of highly conserved amino acids within the cysteine loop have been postulated to play a role in agonist binding. Here, using site-directed mutagenesis and oocyte expression, we have examined the effects of mutating amino acids comprising the cysteine loop on the activation of recombinant G ABA channels composed of rat alpha 1, beta 2, and gamma 2 subunits. Pr eventing the formation of the putative cysteine-cysteine disulfide bon d in any of the subunits, by mutating the cysteine at position 15 to s erine, prevented the functional expression of that subunit. For exampl e, coexpression of gamma(C15S) with wild-type alpha and beta subunits resulted in GABA-activated currents with properties identical to those of GABA-activated currents from coexpression of alpha and beta subuni ts alone. These properties included sensitivity to activation by GABA (similar EC(50) values), blockade by Zn2+, and lack of modulation by t he benzodiazepine diazepam. We also mutated conserved amino acids in t he p subunit that had been specifically proposed to form the GABA bind ing site (beta(R6), beta(Y8), and beta(D11)). These mutations (as well as several others within or adjacent to the cysteine loop) produced e ither a very moderate effect or no effect on GABA sensitivity, suggest ing that these particular amino acids do not play a key role in activa tion of the GABA channel. The data presented in this study support a r ole for the cysteine loop in subunit assembly, rather than channel act ivation.